6 research outputs found
Characterisation of the molecular mechanism required for glucocorticoid augmentation of macrophage phagocytosis of apoptotic neutrophils
The successful resolution of inflammation requires removal of neutrophils from the
inflammatory site to prevent release of histotoxic contents that may potentiate
inflammatory processes and promote progression to a chronic state associated with
impaired repair mechanisms and/or autoimmune responses. Macrophages are
“professional” phagocytes required for rapid and efficient clearance of apoptotic
neutrophils. Macrophage phagocytic capacity can be critically regulated by a number
of environmental factors, including cytokines, bacterial products, and glucocorticoids.
We have hypothesised that modulation of macrophage phagocytic capacity may
represent an effective strategy for promoting resolution of inflammation in diseases
where clearance of neutrophils may be impaired or inefficient. The aim of this thesis
was to investigate the molecular mechanisms underlying glucocorticoid-augmentation
of macrophage phagocytosis. We have demonstrated that long-term exposure of
human peripheral blood monocytes to the synthetic glucocorticoid dexamethasone
dramatically increases phagocytic capacity for “early” membrane-intact apoptotic
neutrophils. Increased phagocytic potential was associated with a “switch” from a
serum-independent to a serum-dependent apoptotic cell recognition mechanism. We
initially employed an “add back” approach to rule out several well-defined opsonins in
apoptotic neutrophil clearance, including immune complexes, IgG, complement
proteins, pentraxin-3, fibronectin, annexin I, and platelet-derived factors. Using a
multi-step purification scheme involving anion exchange and gel filtration
chromatography, we purified a high molecular weight fraction that contained the prophagocytic
activity of serum and analysis by mass spectrometry identified C4-binding
protein as a candidate protein. C4-binding protein circulates in human plasma bound
predominately in a >570kDa complex with protein S and the presence of protein S in
high molecular weight fractions was confirmed by immunoblotting. We found that
protein S was equivalent to unfractionated serum in its ability to enhance phagocytosis
of apoptotic neutrophils by dexamethasone-treated monocyte-derived macrophages
(Dex-MDMo) and that immunodepletion of protein S resulted in loss of prophagocytic
activity. Protein S was found to opsonise apoptotic neutrophils in a
calcium-dependent manner and enhanced phagocytic potential by Dex-MDMo through stimulation of Mer tyrosine kinase (Mertk), a receptor that is upregulated on
the surface of Dex-MDMo compared to untreated MDMo.
The studies presented in this thesis have provided novel insight into the underlying
molecular mechanisms required for high capacity clearance of apoptotic neutrophils
by macrophages following treatment with glucocorticoids and may form the
foundations for further studies investigating glucocorticoid action for development of
safer and more selective therapies
Glucocorticoids Induce Protein S-Dependent Phagocytosis of Apoptotic Neutrophils by Human Macrophages
Characterisation of the molecular mechanism required for glucocorticoid augmentation of macrophage phagocytosis of apoptotic neutrophils
The successful resolution of inflammation requires removal of neutrophils from the inflammatory site to prevent release of histotoxic contents that may potentiate inflammatory processes and promote progression to a chronic state associated with impaired repair mechanisms and/or autoimmune responses. Macrophages are “professional” phagocytes required for rapid and efficient clearance of apoptotic neutrophils. Macrophage phagocytic capacity can be critically regulated by a number of environmental factors, including cytokines, bacterial products, and glucocorticoids. We have hypothesised that modulation of macrophage phagocytic capacity may represent an effective strategy for promoting resolution of inflammation in diseases where clearance of neutrophils may be impaired or inefficient. The aim of this thesis was to investigate the molecular mechanisms underlying glucocorticoid-augmentation of macrophage phagocytosis. We have demonstrated that long-term exposure of human peripheral blood monocytes to the synthetic glucocorticoid dexamethasone dramatically increases phagocytic capacity for “early” membrane-intact apoptotic neutrophils. Increased phagocytic potential was associated with a “switch” from a serum-independent to a serum-dependent apoptotic cell recognition mechanism. We initially employed an “add back” approach to rule out several well-defined opsonins in apoptotic neutrophil clearance, including immune complexes, IgG, complement proteins, pentraxin-3, fibronectin, annexin I, and platelet-derived factors. Using a multi-step purification scheme involving anion exchange and gel filtration chromatography, we purified a high molecular weight fraction that contained the prophagocytic activity of serum and analysis by mass spectrometry identified C4-binding protein as a candidate protein. C4-binding protein circulates in human plasma bound predominately in a >570kDa complex with protein S and the presence of protein S in high molecular weight fractions was confirmed by immunoblotting. We found that protein S was equivalent to unfractionated serum in its ability to enhance phagocytosis of apoptotic neutrophils by dexamethasone-treated monocyte-derived macrophages (Dex-MDMo) and that immunodepletion of protein S resulted in loss of prophagocytic activity. Protein S was found to opsonise apoptotic neutrophils in a calcium-dependent manner and enhanced phagocytic potential by Dex-MDMo through stimulation of Mer tyrosine kinase (Mertk), a receptor that is upregulated on the surface of Dex-MDMo compared to untreated MDMo. The studies presented in this thesis have provided novel insight into the underlying molecular mechanisms required for high capacity clearance of apoptotic neutrophils by macrophages following treatment with glucocorticoids and may form the foundations for further studies investigating glucocorticoid action for development of safer and more selective therapies.EThOS - Electronic Theses Online ServiceGBUnited Kingdo
Glucocorticoids induce protein S-dependent phagocytosis of apoptotic neutrophils by human macrophages
During resolution of an inflammatory response, recruited neutrophil granulocytes undergo apoptosis and are removed by tissue phagocytes before induction of secondary necrosis without provoking proinflammatory cytokine production and release. Promotion of physiological neutrophil clearance mechanisms may represent a viable therapeutic strategy for the treatment of inflammatory or autoimmune diseases in which removal of apoptotic cells is impaired. The mechanism underlying enhancement of macrophage capacity for phagocytosis of apoptotic cells by the powerful anti-inflammatory drugs of the glucocorticoid family has remained elusive. In this study, we report that human monocyte-derived macrophages cultured in the presence of dexamethasone exhibit augmented capacity for phagocytosis of membrane-intact, early apoptotic cells only in the presence of a serum factor. Our results eliminate a role for a number of potential opsonins, including complement, pentraxin-3, and fibronectin. Using ion-exchange and gel filtration chromatography, we identified a high molecular mass serum fraction containing C4-binding protein and protein S responsible for the augmentation of phagocytosis of apoptotic neutrophils. Because the apoptotic neutrophils used in this study specifically bind protein S, we suggest that glucocorticoid treatment of macrophages induces a switch to a protein S-dependent apoptotic cell recognition mechanism. Consistent with this suggestion, pretreatment of macrophages with Abs to Mer tyrosine kinase, a member of the Tyro3/Axl/Mer family of receptor tyrosine kinases, prevented glucocorticoid augmentation of phagocytosis. Induction of a protein S/Mer tyrosine kinase-dependent apoptotic cell clearance pathway may contribute to the potent anti-inflammatory effects of glucocorticoids, representing a potential target for promoting resolution of inflammatory responses